Robert F. Graziano

Clinical significance of IgG Fc receptors and FcγR-directed immunotherapies

Abstract Fc receptors for IgG (FcγRs) can trigger the inflammatory, cytotoxic and hypersensitivity functions of immune effector cells. Activation or deactivation of effector cells via FcγRs can be exploited to develop novel therapies for cancer, infectious diseases and autoimmune disorders. Initial results of clinical trials for several FcγR-directed immunotherapies show the potential promise of this approach.

Human Monoclonal Antibodies Directed against Toxins A and B Prevent Clostridium difficile-Induced Mortality in Hamsters

ABSTRACT Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P < 0.0001) in the primary disease hamster model and from 78% to 32% (P < 0.0001) in the less stringent relapse model.

Antigen targeting to myeloid-specific human Fc gamma RI/CD64 triggers enhanced antibody responses in transgenic mice.

Besides their phagocytic effector functions, myeloid cells have an essential role as accessory cells in the induction of optimal humoral immune responses by presenting captured antigens and activating lymphocytes. Antigen presentation by human monocytes was recently found to be enhanced in vitro through the high-affinity Fc receptor for IgG (Fc gamma RI; CD64), which is exclusively present on myeloid cells. To evaluate a comparable role of Fc gamma RI in antigen presentation in vivo, we generated human Fc gamma RI transgenic mice. Under control of its endogenous promoter, human Fc gamma RI was selectively expressed on murine myeloid cells at physiological expression levels. As in humans, expression was properly regulated by the cytokines IFN-gamma, G-CSF, IL-4, and IL-10, and was up-regulated during inflammation. The human receptor expressed by murine macrophages bound monomeric human IgG and mediated particle phagocytosis and IgG complex internalization. To evaluate whether specific targeting of antigens to Fc gamma RI can induce enhanced antibody responses, mice were immunized with an anti-human Fc gamma RI antibody containing antigenic determinants. Transgenic mice produced antigen-specific antibody responses with high IgG1 titers and substantial IgG2a and IgG2b responses. These data demonstrate that human Fc gamma RI on myeloid cells is highly active in mediating enhanced antigen presentation in vivo, and show that anti-Fc gamma RI mAbs are promising vaccine adjuvants.

Increased potency of Fc-receptor-targeted antigens

Abstract A major challenge for using native and modified T cell epitopes to induce or suppress immunity relates to achieving efficient uptake and processing by antigen-presenting cells (APC) in vivo. IgG Fc receptors, which are expressed constitutively by professional APC including monocytes and dendritic cells, have long been known to mediate antigen uptake in a manner leading to efficient T cell activation. We have previously demonstrated enhanced presentation of antigenic and antagonistic peptides by targeting them to the type I Fc receptor for IgG (FcγRI, CD64) on human monocytes. In the present report we review the literature suggesting that CD64-targeted antigens are likely to be effective in vivo, and present data demonstrating enhanced immunogenicity in CD64 transgenic mice of a fusion protein that combines the specificities of HIV gp120 and the humanized anti-CD64 monoclonal antibody H22. Overall, these studies suggest that targeting antigens to CD64 represents an effective approach to enhancing the effectiveness of vaccines in vivo.

Activity and Safety of CTLA-4 Blockade Combined with Vaccines in Cynomolgus Macaques

The immune modulatory molecule CTLA-4 (CD152), through interactions with the B7 costimulatory molecules, has been shown to be a negative regulator of T cell activation in various murine model systems. Abs that block CTLA-4 function can enhance immune responses that mediate potent antitumor activity. However, CTLA-4 blockade can also exacerbate autoimmune disease. The safety and activity of anti-CTLA-4 Abs in primates has not been addressed. To that end, we generated human Abs against CTLA-4 using transgenic mice expressing human Ig genes. A high affinity Ab (10D1) that blocked the binding of CTLA-4 to the B7-1 and B7-2 ligands and had cross-reactivity with macaque CTLA-4 was chosen for further development. Administration of 10D1 to cynomolgus macaques significantly enhanced Ab responses to hepatitis surface Ag and a human melanoma cell vaccine. Anti-self Ab responses as measured by immunoassays using lysate from melanocyte-rich tissues were elicited in those animals receiving the melanoma cell vaccine and anti-CTLA-4 Ab. Remarkably, chronic administration of 10D1 did not result in measurable polyclonal T cell activation, significant alteration of the lymphocyte subsets, or induce clinically observable autoimmunity. Repeated dosing of the 10D1 did not elicit monkey anti-human Ab responses in the monkeys. These observations support the development of CTLA-4 blockade for human immunotherapy.

Triggering FCα-Receptor I (CD89) Recruits Neutrophils as Effector Cells for CD20-Directed Antibody Therapy

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In 51Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcαRI) and CD20, a broad range of B cell lines were effectively killed. FcαRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcαRI × CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcγRI- or FcγRIII-directed BsAbs against CD20. Experiments with blood from human FcαRI/FcγRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcαRI × CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.

Development and Characterization of a Severe Acute Respiratory Syndrome—Associated Coronavirus—Neutralizing Human Monoclonal Antibody That Provides Effective Immunoprophylaxis in Mice

Abstract Background. Severe acute respiratory syndrome (SARS) remains a significant public health concern after the epidemic in 2003. Human monoclonal antibodies (MAbs) that neutralize SARS-associated coronavirus (SARSCoV) could provide protection for exposed individuals. Methods. Transgenic mice with human immunoglobulin genes were immunized with the recombinant major surface (S) glycoprotein ectodomain of SARS-CoV. Epitopes of 2 neutralizing MAbs derived from these mice were mapped and evaluated in a murine model of SARS-CoV infection. Results. Both MAbs bound to S glycoprotein expressed on transfected cells but differed in their ability to block binding of S glycoprotein to Vero E6 cells. Immunoprecipitation analysis revealed 2 antibody-binding epitopes: one MAb (201) bound within the receptor-binding domain at aa 490–510, and the other MAb (68) bound externally to the domain at aa 130–150. Mice that received 40 mg/kg of either MAb prior to challenge with SARS-CoV were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection. Conclusions. Two neutralizing epitopes were defined for MAbs to SARS-CoV S glycoprotein. Antibodies to both epitopes protected mice against SARS-CoV challenge. Clinical trials are planned to test MAb 201, a fully human MAb specific for the epitope within the receptor-binding region.

F(c)gammaRI-targeted fusion proteins result in efficient presentation by human monocytes of antigenic and antagonist T cell epitopes.

A major challenge for using native or modified T cell epitopes to induce or suppress immunity relates to poor localization of peptides to antigen presenting cells (APCs) in vivo. In this study, we demonstrate enhanced presentation of antigenic and antagonistic peptides by targeting them to the type I Fc receptor for IgG (F(c)gammaRI, CD64) on human monocytes. A Th epitope of tetanus toxoid, TT830, and the antagonistic peptide for TT830, TT833S, were genetically grafted into the constant region of the heavy chain of the humanized anti-CD64 mAb 22 and expressed as monovalent fusion proteins, Fab22-TT830 and Fab22-TT833S. These CD64-targeted peptides were up to 1,000- and 100-fold more efficient than the parent peptides for T cell stimulation and antagonism, respectively, suggesting that such fusion proteins could effectively increase the delivery of peptides to APCs in vivo. Moreover, the F(c)gammaRI-targeted antagonistic peptide inhibited proliferation of TT830-specific T cells even when APCs were first pulsed with native peptide, a situation comparable with that which would be encountered in vivo when attempting to ameliorate an autoimmune response. These data suggest that targeted presentation of antagonistic peptides could lead to promising Ag-specific therapies for T cell-mediated autoimmune diseases.

Differential Effect of Cytokine Treatment on Fcα Receptor I- and Fcγ Receptor I-Mediated Tumor Cytotoxicity by Monocyte-Derived Macrophages

Macrophages represent an important effector cell for Ab-mediated tumor therapy. Previous studies have documented that cytokines can influence Fc receptor (FcR) expression and function. In this study we examined the tumoricidal activities of the type I receptors for IgG (FcγRI) and the IgA FcR (FcαRI) on monocyte-derived macrophages (MDM) cultured in the presence of IFN-γ, M-CSF, or GM-CSF. Bispecific Abs were used to target a Her2/neu breast carcinoma cell line, SKBR-3, to FcαRI or FcγRI on MDM. Although FcαRI and FcγRI share a common signaling pathway contingent on association with the γ-chain (FcRγ subunit), a marked difference in their efficiency in mediating tumoricidal functions was seen in response to specific cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient phagocytosis of SKBR-3 cells by FcαRI and FcγRI; however, IFN-γ-treated MDM phagocytosed tumor cells only with the FcγRI-directed bispecific Abs. Similarly, IFN-γ-cultured MDM lysed tumor cells more efficiently via FcγRI then by FcαRI as measured in Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated MDM mediated more efficient lysis of tumor cells via FcαRI than FcγRI, while M-CSF-cultured MDM were relatively less efficient in mediating Ab-dependent cellular cytotoxicity through either receptor. With the exception of IFN-γ-mediated enhancement of FcγRI expression and FcγRI γ-chain complexes, the regulation of FcγRI- or FcαRI-mediated activity occurred without significant change in either receptor expression or total complexes with γ subunit. These data suggest that the efficiency of Ab-mediated tumor therapy, which depends on FcR effector cell functions, may be modified by the use of specific cytokines.

Targeting Weak Antigens to CD64 Elicits Potent Humoral Responses in Human CD64 Transgenic Mice

Previous studies have documented that targeting foreign Ags to IgG FcγR leads to enhanced Ag-specific responses in vitro and in vivo. However, the ability to overcome immunologic nonresponsiveness by targeting poorly immunogenic Ags to FcγR has not been investigated. To address this question in a simple model, we immunized transgenic mice expressing human CD64 (FcγRI) and their nontransgenic littermates with Fab′ derived from the murine anti-human CD64 mAb m22. The m22 Fab′ served as both the targeting molecule and the Ag. We found that only CD64-expressing mice developed anti-Id titers to m22. Furthermore, chemically linked multimers of m22 Fab′, which mediated efficient internalization of the human CD64, were significantly more potent than monomeric m22 F(ab′)2 at inducing anti-Id responses. In all cases, the humoral responses were specific for m22 Id and did not react with other murine IgG1 Fab′ fragments. Chemical addition of a second murine Fab′ (520C9 anti-human HER2/neu) to m22 Fab′ multimers demonstrated that IgG1 and IgG2a anti-Id titers could be generated to 520C9 only in the CD64-expressing mice. These results show that targeting to CD64 can overcome immunological nonresponsiveness to a weak immunogen. Therefore, targeting to CD64 may be an effective method to enhance the activity of nonimmunogenic tumor vaccines.